Functional dissection of the proton pumping modules of mitochondrial complex I.
Functional dissection of the proton pumping modules of mitochondrial complex I.
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DOI：
10.1371/journal.pbio.1001128
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发表时间：
2011-08
期刊：
影响因子：
9.8
通讯作者：
Brandt U
中科院分区：
文献类型：
--
作者：
Dröse S;Krack S;Sokolova L;Zwicker K;Barth HD;Morgner N;Heide H;Steger M;Nübel E;Zickermann V;Kerscher S;Brutschy B;Radermacher M;Brandt U
呼吸链复合体I的催化活性亚复合物缺少42个亚基中的14个，但仍保留了一半的质子泵送能力，表明其膜臂具有两个泵送模块。线粒体复合体I是呼吸链中最大和最复杂的质子泵，它将NADH到泛醌的电子传递与从基质向膜间空间的四个质子的泵送联系起来。在人类中，复合体I的缺陷与多种退行性疾病有关。原核和真核复合体I的x射线结构分析的最新进展证实，氧化还原反应完全局限于l型分子的亲水外周臂，并且发生在离膜结构域相当远的地方。虽然这清楚地表明，配合物I膜臂内的质子泵浦是通过远程构象耦合间接驱动的，但分子机制以及泵浦位点的数量、身份和定位仍不清楚。在这里，我们报道，在删除耶氏复合体I的一个小附属亚基的基因后，形成了一个稳定的亚复合体（nb8mΔ），缺乏膜结构域的远端部分，如单颗粒分析所示。通过三种互补的蛋白质组学方法对holo和亚复合物的亚基组成进行分析，发现在nb8mΔ中缺失了与细菌mrp型Na+/H+反转运蛋白同源的两个亚基（ND4和ND5），这些亚基被认为是质子泵的主要候选。尽管如此，nb8mΔ仍然以整个酶一半的化学计量量抽吸质子。我们的研究结果提供了证据，证明复合物I的膜臂含有两个功能不同的泵模块，它们通过x射线结构分析最近发现的长螺旋传输元件串联在一起。线粒体——真核细胞的发电厂——以ATP的形式产生能量。超过三分之一的能量产生是由质子穿过线粒体膜的梯度驱动的，这种梯度是由一种叫做复合体I的非常大的酶的泵送作用产生的。复合体I的缺陷涉及许多病理过程，如神经变性和生物衰老。最近的x射线结构分析显示，复合体I是一个l形分子，其中一只手臂集成到膜上，另一只手臂粘在线粒体的水内部；酶的化学反应发生在亲水性臂上，与质子抽运明显分开，质子抽运必须发生在膜臂的某个地方。为了将泵功能分配到结构域，我们通过删除酵母中的一个小亚基的编码基因，创建了一个稳定的复合体I亚复合体。这个亚复合物缺少一半的膜臂；它仍然具有催化活性，但它所抽吸的质子数量只有完整络合物的一半。这表明复合体I在其膜臂中有两个功能不同的泵模块。
A catalytically active subcomplex of respiratory chain complex I lacks 14 of its 42 subunits yet retains half of its proton-pumping capacity, indicating that its membrane arm has two pump modules. Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8mΔ) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na+/H+ antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8mΔ. Nevertheless, nb8mΔ still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis. Mitochondria—the power plants of eukaryotic cells—produce energy in the form of ATP. More than one-third of this energy production is driven by a gradient of protons across the mitochondrial membrane created by the pumping action of a very large enzyme called complex I. Defects in complex I are implicated in numerous pathological processes like neurodegeneration and biological aging. Recent X-ray structural analyses revealed that complex I is an L-shaped molecule with one arm integrated into the membrane and the other sticking into the aqueous interior of the mitochondrion; the chemical reactions of the enzyme take place in this hydrophilic arm, clearly separated from proton pumping that must occur somewhere in the membrane arm. To assign the pump function to structural domains, we created a stable subcomplex of complex I by deleting the gene encoding one of its small subunits in a yeast called Yarrowia lipolytica. This subcomplex lacked half of the membrane arm; it was still catalytically active but it pumped only half the number of protons as the full complex. This indicates that complex I has two functionally distinct pump modules operating in its membrane arm.
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